Hypertrophic cardiomyopathy (HCM) affects 1 in 500 people in the general population and is a leading cause of heart failure and sudden cardiac death. Despite the significant clinical impact and ongoing basic and translational research, the molecular mechanisms leading to disease onset and progression are poorly understood. Until now, no specific therapeutic approach has been established. In vitro modeling of cardiovascular diseases is of high interest as human adult cardiomyocytes are difficult to isolate and propagate long-term in culture, and animal models have often proven non-predictive of human pathophysiology. Patient- specific human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and genome editing represent novel technologies for modeling cardiomyopathies. In the past funding period, we were able to recruit ~150 HCM patients. Preliminary data shows diastolic dysfunction and aberrant calcium handling in iPSC-CMs with mutations in myosin binding protein C3 (MYBPC3). Furthermore, preliminary data shows a molecular phenotype in MYBPC3 iPSC-CMs characterized by an activation of non-sense mediated decay (NMD) pathway. However, a major limitation of iPSC-CMs is their immature state as well as the non- physiological conditions of in vitro assays used so far. Here we propose to increase the significance of modeling HCM by i) incorporating 2D micropatterned iPSC-CMs and 3D engineered heart tissues (EHTs) to enhance cellular maturation; by ii) co-culturing iPSC-CMs with iPSC-derived endothelial cells (iPSC-ECs) and cardiac fibroblasts (iPSC-CFs) in 3D EHTs, and by (iii) using single cell RNA-seq and proteomics approaches to elucidate molecular mechanisms of HCM cellular crosstalk. Finally, we will evaluate the significance of the NMD pathway in HCM pathogenesis by using the CRISPR-interference (CRISPRi) and CRISPR-activation (CRISPa) technology, which allows us to screen expeditiously the functional relevance of inhibition or activation of a set of genes followed by downstream functional evaluation to localize the target genes with therapeutic potentials.

Public Health Relevance

Cardiomyocytes from human induced pluripotent stems cells (iPSC-CMs) are a powerful tool to understand hypertrophic cardiomyopathy (HCM). This study will use genome-edited isogenic iPSC-CMs to investigate the molecular mechanisms on how MYBPC3 mutations lead to disease onset. We will use recent advances in 2D micropatterning and 3D engineering to promote cellular maturation in vitro. We will evaluate cellular crosstalk through co-culture of different iPSC-derived cardiovascular cell types (i.e., cardiomyocytes, endothelial cells, and cardiac fibroblasts) by proteomics and single cell sequencing. Finally, we will evaluate the significance of the non-sense mediated decay (NMD) targeted downstream pathways in the pathogenesis of MYBPC3 HCM, and screen for novel therapeutic targets using CRISPRi/a technology.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL126527-06
Application #
9971569
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Desvigne-Nickens, Patrice
Project Start
2015-03-10
Project End
2023-06-30
Budget Start
2020-07-01
Budget End
2021-06-30
Support Year
6
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Stanford University
Department
Radiation-Diagnostic/Oncology
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94305
Chang, Alex C Y; Chang, Andrew C H; Kirillova, Anna et al. (2018) Telomere shortening is a hallmark of genetic cardiomyopathies. Proc Natl Acad Sci U S A 115:9276-9281
Lee, Jaecheol; Shao, Ning-Yi; Paik, David T et al. (2018) SETD7 Drives Cardiac Lineage Commitment through Stage-Specific Transcriptional Activation. Cell Stem Cell 22:428-444.e5
Knowles, Joshua W; Ashley, Euan A (2018) Cardiovascular disease: The rise of the genetic risk score. PLoS Med 15:e1002546
Zhang, Joe Z; Guo, Hongchao; Wu, Joseph C (2018) Applications of genetically engineered human pluripotent stem cell reporters in cardiac stem cell biology. Curr Opin Biotechnol 52:66-73
Rhee, Siyeon; Chung, Jae I; King, Devin A et al. (2018) Endothelial deletion of Ino80 disrupts coronary angiogenesis and causes congenital heart disease. Nat Commun 9:368
Ma, Ning; Zhang, Joe Z; Itzhaki, Ilanit et al. (2018) Determining the Pathogenicity of a Genomic Variant of Uncertain Significance Using CRISPR/Cas9 and Human-Induced Pluripotent Stem Cells. Circulation 138:2666-2681
Liu, Chun; Oikonomopoulos, Angelos; Sayed, Nazish et al. (2018) Modeling human diseases with induced pluripotent stem cells: from 2D to 3D and beyond. Development 145:
Rhee, June-Wha; Wu, Joseph C (2018) Cardiac Cell Cycle Activation as a Strategy to Improve iPSC-Derived Cardiomyocyte Therapy. Circ Res 122:14-16
Shukla, Praveen; Wu, Joseph C (2017) Cardiac subtype characterization using all-optical action potential imaging. Eur Heart J 38:302-304
Churko, Jared M; Lee, Jaecheol; Ameen, Mohamed et al. (2017) Transcriptomic and epigenomic differences in human induced pluripotent stem cells generated from six reprogramming methods. Nat Biomed Eng 1:826-837

Showing the most recent 10 out of 33 publications